puber, Cancer pagurus, Uca pugilator and Maja squinado. They were only observed in the X-organ sinus gland (SG) system of the eyestalks and consisted of ...
Cell Tissue Res (1988) 251:3-12
a n d Tissue Research 9 Springer-Verlag 1988
Immunocytochemical demonstration of the neurosecretory systems containing putative moult-inhibiting hormone and hyperglycemic hormone in the eyestalk of brachyuran crustaceans Heinrich Dircksen 1, Simon G. Webster 2 and Rainer Keller 1 1 Rheinische Friedrich-Wilhelms Universit~it, Institut ffir Zoophysiologie, Bonn, Federal Republic of Germany; 2 University of North Wales, School of Animal Biology, Bangor, Gwynedd, United Kingdom
Summary. By use of antisera raised against purified moultinhibiting (MIH) and crustacean hyperglycemic hormone (CHH) from Carcinus maenas, complete and distinct neurosecretory pathways for both hormones were demonstrated with the PAP and immunofluorescence technique. By double staining, employing a combination of silver-enhanced immunogold labelling and PAP, both antigens could be visualized in the same section. Immunoreactive structures were studied in Carcinus maenas, Liocareinus puber, Cancer pagurus, Uca pugilator and Maja squinado. They were only observed in the X-organ sinus gland (SG) system of the eyestalks and consisted of MIH-positive perikarya, which were dispersed among the more numerous CHH-positive perikarya of the medulla terminalis X-organ (XO). The MIH-positive neurons form branching collateral plexuses adjacent to the XO and axons that are arranged around the CHH-positive central axon bundle of the principal XO-SG tract. In the SG, MIH-positive axon profiles and terminals, clustered around hemolymph lacunae, are distributed between the more abundant CHH-positive axon profiles and terminals. Colocalisation of M I H and C H H was never observed. The gross morphology of both neurosecretory systems was similar in all species examined, however, in U. pugilator and M. squinado immunostaining for M I H was relatively faint unless higher concentrations of antiserum were used. Possible reasons for this phenomenon as well as observed moult cycle-related differences in immunostaining are discussed. Key words: Moult-inhibiting hormone - Hyperglycemic hormone - Immunocytochemistry - Neurosecretion - Decapod crustaceans (five species)
Skinner 1985a, b; Webster and Keller, 1987, for reviews). Whilst other factors may also be involved in the various physiological processes related to moult cycle integration (Skinner 1985b), recent research using in vivo (Keller and O'Connor 1982; Bruce and Chang 1984; Snyder and Chang 1986), and in vitro (Soumoff and O'Connor 1982; Mattson and Spaziani 1985, Webster 1986) bioassays for M I H have repeatedly affirmed this simple model for moult control. In particular, the Y-organ in vitro assay for M I H has allowed complete purification and amino acid analysis of a putative M I H in Carcinus maenas (Webster and Keller 1986). Since these studies demonstrated that M I H was one of the major components of the neuropeptide inventory of the sinus gland in Carcinus maenas, it was feasible to raise an antiserum directed against M I H of C. maenas to elucidate the neurosecretory pathways of this peptide by immunocytochemical techniques. In this paper we report, for the first time, the immunocytochemical localisation of the M I H neurosecretory system in several species of brachyuran crustaceans. At an early stage, this study revealed that the somata of the MIH-immunopositive neurons are located in the medulla-terminalis X-organ (XO), which, as former investigations had shown (Keller et al. 1985), also contains the very conspicuous group of perikarya producing crustacean hyperglycemic hormone (CHH). Moreover, the MIH-perikarya seemed indistinguishable in size and shape from the CHH-cells. It was therefore mandatory to include the latter in this study to determine whether both neuropeptides were associated with distinct populations of neurons or whether colocalisation occurred. Due to the availability of highly specific antisera, it was possible to show that M I H and C H H are associated with morphologically similar but clearly distinct neurosecretory pathways. Materials and methods
The most widely accepted hypothesis concerning the control of moulting in crustaceans proposes that an increase in ecdysteroid synthesis and consequently haemolymph titre necessary to initiate and sustain proecdysis, and ultimately moulting, is regulated by a moult-inhibiting hormone (MIH) which is produced by the eyestalk neurosecretory complex. The main action of this hormone is to repress ecdysteroid synthesis in the Y-organs during intermoult (see Send offprint requests to: H. Dircksen, Institut ffir Zoophysiologie der Universitfit, Endenicher Allee 11-13, D-5300 Bonn, Federal Republic of Germany
(a) Animals and tissue preparation. Carcinus maenas were obtained from the Nederlands Instituut voor bet Onderzoek van de Zee, Texel, Netherlands, or from the shore of Anglesey, Wales, UK, and were maintained in filtered, recirculating artificial seawater at 9 ~ C, under a photoperiodic regime of 18 L 6D. Liocareinus puber, Cancer pagurus and terminally anecdysic Maja squinado were collected by local fishermen and maintained and prepared at the Laboratoire de Biologie Marine, Coll6ge de France, Concarneau, France. Uca pugilator, from the shore near Pensacola, Florida, USA, were maintained as described previously (Keller
1977). Preparations were always made during the day, and most animals were in intermoult stage C4, although some were examined during late premoult (Stage D2). Eyestalk ganglia were dissected under ice-cold saline (Nordmann and Morris 1980) and were immediately fixed in one of the following solutions: (a) Bouin's fluid, 4-24 h at room temperature (RT), (b) Bouin's fluid without acetic acid 4-24 h at RT, (c) Boer's fixative (Boer et al. 1979), (d) Stefanini's fixative (Stefanini et al. 1967). Embedding was carried out in Paraplast after dehydration in graded ethanol series via methylbenzoate-benzene.
(b) Peptide pur(/i'cation and antisera production. MIH and CHH were isolated from sinus glands (SG) of Carcinus maenas. MIH was purified by high pressure liquid chromatography (HPLC) of acetic acid extracts (2N) of SGs as described by Webster and Keller (1986). CHH, extracted in 0.05 M ammonium acetate buffer (pH 8.5), was pre-purifled by gel filtration on a SephadexG 50 superfine column (Keller 1981) and isolated by HPLC according to Keller and Kegel (1984). Both hormones were quantified by automated amino acid analyses on a LC 5000 amino acid analyzer (Biotronik, Munich, FRG) after hydrolysis in constant boiling HC1 (Pierce) for 24 h at 110~ C. Two New Zealand white rabbits ( ~ 2 kg) were each injected sub- and intracutaneously with 70 lag MIH, dissolved in 0.75 ml 0.1 M phosphate buffer (pH 7.4), emulsified with 0.75 ml Freund's complete adjuvant (DIFCO). After 31 days, a booster injection of 35 gg MIH/animal was given in the same way, but using Freund's incomplete adjuvant (DIFCO). Several ear bleedings were carried out to measure rise in antibody titre (by RIA of MIH, Webster et al., in preparation). The rabbits were terminally exsanguinated under anaesthesia 68 days after the booster injections. In the case of CHH, two rabbits ( ~ 2.5 kg) each received 125 lag CHH, followed by a single booster of 75 lag CHH 35 days later, using the same protocol as described for MIH. Antibody titres were checked by an improved modification (Keller 1987) of the RIA for CHH, originally described by Jaros and Keller (1979 b). These rabbits were terminally exsanguinated under anaesthesia 28 days after the booster injection. Plasma fractions from retracted blood clots were stored at - 2 0 ~ C. ( c ) Immunoc ytochemistry and specificity controls. Paraplast sections (5 7 jam, occasionally 12 lam) were immunostained by the peroxidase-antiperoxidase (PAP) technique (Sternberger 1979), or by an immunogold-silver method with selfprepared goat-anti-rabbit-IgG 40-nm colloidal gold conjugates as described elsewhere (Dircksen et al. 1987). Double staining for both antigens was performed by sequential immunogold-silver staining and PAP with an intermediate antibody elution step in 0.1 M glycine-HC1 buffer (pH 2.2) for 1 h at 37 ~ C or 2 h at RT as modified after Gu et al. (1981). Sections were incubated for 1 h at 37 ~ or overnight at 4 ~ with primary antisera diluted 1:4000-1:10000 for the anti-MIH serum (code RIB3 or R1TB) and 1:6000-1:12000 for the anti-CHH serum (code RIBI/3) with 0.01 M phosphate-buffered saline (PBS), (pH 7.4). Indirect immunofluorescence technique was applied to sections or whole-mount preparations of eyestalk ganglia. The latter were fixed according to Stefanini et al. (1967) for 6 - 1 2 h at 4~ washed in 0.1 M Na-phosphate buffer (pH 7.4) containing 0.8 M sucrose (3 x 10 rain, 4 ~ C), the
same buffer containing 0.5% Triton X 100 (3 x 10 min, 4 ~ C), and finally three times in 0.1 M PBS containing 0.5% Triton X-100 (PTX) (10-30 rain, 4 ~ C). Incubations in primary antiserum diluted 1:500-1 : 1000 were performed overnight at 4 ~ C, followed by extensive washing in PTX, and incubation in FITC-coupled goat-anti-rabbit-IgG (Sigma) diluted ! :30 in PTX for 1-2 h at RT. Sections or wholemount preparations were mounted in glycerol/PTX (1:1) and viewed under a Leitz Orthoplan/Ploemopak 2.1 fluorescence microscope equipped with an Hg-epi-illumination light source (HBO 200 W/4) and I2 filter system. Photographs using bright field illumination were also taken with the same microscope. The specificity of the antisera was tested by immunodot blotting of peak fractions after HPLC of sinus gland extracts as described by Dircksen et al. (1987), Western blots after SDS-gel electrophoresis (Towbin et al. 1979), and by preabsorption controls on sections. Normally, about 10 lag MIH or CHH per lal of antiserum were sufficient to block specific staining completely. In the case of MIH the PBS for antiserum dilution contained 450 mMol/l NaC1 to increase the solubility of the hormone. Results
All rabbits produced antisera of high titre against the HPLC-purified hormones, permitting clear, backgroundfree staining on sections at dilutions higher than 1 : 10000. Their specificity was restricted to the two known peak fractions of MIH or CHH after HPLC of SG extracts, as demonstrated by immuno-dot blotting (Fig. 1). In Western blots, the antisera stained only single bands, corresponding to CHH and MIH, respectively (data not shown). Immunocytochemical investigations of single and serial sections revealed two distinct systems of neurosecretory cells in the X organ-sinus gland complex. No MIH or CHH immunoreactivity was observed in the cerebral or thoracic ganglia, including all connectives and commissures. Different fixation regimes produced essentially similar results, but increased background staining was often observed after fixation with Boer's fixative. The distinct staining of medulla terminalis X-organ (XO)-perikarya or axons, and axon profiles and terminals in the sinus gland (SG) for either MIH or CHH is clearly demonstrated by double staining with silver-enhanced immunogold and PAP, respectively (Fig. 2a, b). Many of the XO-perikarya, measuring 35-45 lam in diameter, are CHH-immunoreactive, directing a large and conspicuous axon bundle to the SG, where a large proportion of the axon profiles and terminals stain positively for CHH. Characteristically, the CHH axons in the SG possess large Herring body-like swellings at the periphery of the SG and terminate with small branches adjacent to the central hemolymph lacunae (Fig. 3c). The MIH-immunoreactire perikarya are less numerous and are localized more peripherally in the XO, but are also intermingled between the CHH-immunoreactive perikarya. They are indistinguishable from the latter with regard to their size and shape. Axons from MIH-immunoreactive perikarya follow, and are arranged circumferentially around the CHH-immunopositive axon bundle (Fig. 2b, inset; Fig. 3i). Normally, some of the MIH-immunoreactive axons run across others before entering the SG (Figs. 3k, 4a). The axons terminate in fine branches with less pronounced Herring body-like
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Fig. 1. HPLC of an 2N acetic acid extract of 40 SG from Carcinus rnaenas. Immunodotting of 40 manually collected peak fractions (5 SG-equivalents/dot) shows only one fraction with MIH- (30) or CHH- (32) immunoreactivity, respectively, at the retention times of isolated hormones (arrows). Gradient elution on a Waters/t-Bondapak-Phenyl column with solvents A: 0.11% TFA and B: 0.10% TFA, 60% CH3CN from 30%-80% B in I h at a flow rate of 0.9 ml/min
axonal swellings and small endings adjacent to the hemolymph lacunae (Figs. 2a, 3a, h). A typical feature of the M I H neurons is the formation of a plexus of axon collaterals in the neuropile o f the medulla terminalis close to the XO (Figs. 2b, 31). A similar plexus is also formed by the CHH-immunopositive neurons (Fig. 4d). In interrnoult C. maenas both the C H H - and MIH-immunoreactive perikarya stained very densely, whereas in late premoult both cell types appeared to display considerably reduced staining associated with granular structures in the cytoplasm of the perikarya. This was particularly pronounced in MIH-immunoreactive cells (Fig. 3e). The staining of both M I H - and CHH-immunoreactive structures is completely abolished by preabsorption of the antisera with the respective antigens, as is shown by use of consecutive sections through the SG of C. maenas (Fig. 3a-d). Staining of consecutive sections through the XO with M I H and C H H antiserum, respectively, demonstrates that both neuropeptides are not colocalised in the perikarya of the XO (Fig. 3 f, g). Although this test has been carried out in C. rnaenas only, the completely different arrangement of M I H - and CHH-neurons indicates lack of co-localisation also in the other species.
A comparative study of the M I H and C H H neurosecretory systems in other brachyurans revealed essentially the same morphology as that seen in C. maenas, particularly in Liocarcinus puber and Cancer pagurus. The arrangement of the M I H perikarya relative to the C H H perikarya in the XO (Fig. 4d, e, i, k), and the peripheral arrangement o f the M I H axons around the C H H axon bundles in the principal XO-SG tract (Fig. 4c, h) were very similar. In the SG, M I H axon profiles and terminals were smaller and less abundant than C H H axon profiles and terminals (Fig. 4a, b, f, g). In both L. puber and C. pagurus, good immunostaining was obtained at the same antiserum concentration as used for C. maenas. In Uea pugilator and Maja squinado, however, much higher concentrations of MIH-antiserum (1:2000-1:4000) were needed to obtain satisfactory immunostaining. In U. pugilator, the M I H - p o s itive perikarya appeared to be of a somewhat larger type than the C H H perikarya (Fig. 5d, e). Compared to the other species, M I H appeared to be more prominent in the SG (Fig. 5a). C H H immunoreactivity in the SG and the XO-SG tract is shown in Fig. 5 b and c. In Maja squinado, M I H terminals in the SG were particularly small and sparse (Fig. 5f), whilst C H H terminals were typically large and
Fig. 2a, b. Double staining by sequential use of immunogold-silver staining for MIH- (black) and PAP for CHH-immunoreactivity (brown) in the SG (a) and the XO (b) of C. maenas. Inset in b) shows a cross section through the XO-SG tract. Note that MIHimmunoreactive neurons form less pronounced Herring body-like swellings of axon profiles in the SG. Axon profiles unreactive with MIH and CHH antiserum indicated by arrowheads in a. (a, b x 290; inset in b x 460). SG sinus gland; XO X-organ: MI, M T medulla interna and terminalis a b u n d a n t (Fig. 5g). A m o n g the species studied, the M I H p e r i k a r y a in the X O a p p e a r e d to c o n t a i n particularly little i m m u n o r e a c t i v e m a t e r i a l in M . squinado (Fig. 5 i), whereas the C H H p e r i k a r y a and their a x o n s displayed very s t r o n g staining (Fig. 5 h, k). By analysis o f serial sections o f eyestalk ganglia, the total n u m b e r o f M I H - a n d C H H - i m m u n o p o s i t i v e cells were e s t i m a t e d ( T a b l e 1). W h i l s t n u m b e r s v a r y a m o n g species,
C H H - i m m u n o p o s i t i v e cells consistently o u t n u m b e r the M I H - i m m u n o p o s i t i v e cells by a ratio o f a p p r o x i m a t e l y 2: 1 in all species examined.
Discussion In the present study, the c o m p l e t e n e u r o s e c r e t o r y p a t h w a y o f the p u t a t i v e m o u l t - i n h i b i t i n g h o r m o n e ( M I H ) o f five
Fig. 3a-I. MIH- and CHH-immunoreactivity in Carcinus maenas, a MIH-positive axon profiles and terminals in the SG. b Control to a, adjacent section treated with preabsorbed antiserum, x 210. e CHH-positive axon profiles and terminals in the SG. d Control to c, adjacent section treated with preabsorbed antiserum, x 180. e MIH-immunoreactive perikarya in the XO and axons in the XO-SG tract (arrows) in the medulla terminalis (MT) in premoult stage De-D3. Note typical, faint and granular staining of the cytoplasm indicative of inactive Nissl substance, x 230. f Section through the XO showing MIH-immunoreactive perikarya and axon collaterals. g Adjacent section stained with CHH-antiserum. Arrows indicate the CHH cells in f and the MIH cells in g. Note lack of co-localisation. x 230. h Section (12 lain) through the SG showing MIH-immunoreactive terminals bordering hemolymph lacuna (asterisks) and Herring body-like thickenings (H). x 560. i Section (12 p,m) through the XO-SG tract in the ventromedial region of the MT. Note densely stained MIH-positive fibres devoid of varicosities, x 310. k Whole-mount preparation of eyestalk ganglia. MlH-immunoreactive axons enter the SG from a medial-lateral position of the MT (large arrow) and form terminals in distal and proximal areas of the SG. Crossing of axons indicated by large arrow, x 140. 1 Whole-mount preparation of the XO. The MIH-immunoreactive perikarya (small arrows) give rise to an axon tract (AT) and a collateral plexus (CP), which extends to the distal surface of the MT ( x 170). For other abbreviations, see Fig. 2. a-g PAP; h-i immunofluorescence
Fig. 3 (For legend see p. 6)
Fig. 4 (For legend see p. 10)
Fig. 5 (For legend see p. 10)
10 Table 1. Numbers of MIH- and CHH-immunoreactive perikarya
in X-organs of five different brachyuran crustaceans Species
MIH-cells
Carcinus maenas Liocarcinus puber Cancer pagurus Uca pugilator Maja squinado
28-36 46 44~48 20-22 n.d.
CHH-cells 62-65 72 75 76 40-46 145
n.d. = not determined
b r a c h y u r a n crustacean species has been described and compared to the crustacean hyperglycemic h o r m o n e (CHH) system. The C H H - c o n t a i n i n g neurosecretory structures of C. maenas have previously been studied by use of an antiserum raised against homogenates of sinus glands (SG) of the same species (Jaros and Keller 1979a). Although the antiserum appeared to contain predominantly antibodies against CHH, the presence of antibodies to MIH, which was unk n o w n at that time, cannot be ruled out. F o r the present study, it was therefore crucial to have antisera that were specific for C H H or MIH, respectively. Such antisera have been produced by use of HPLC-purified peptides as antigens. The M I H - i m m u n o r e a c t i v e system is clearly distinct from the neurosecretory pathways of C H H (Jaros and Keller 1979a; Keller etal. 1985; Gorgels-Kallen etal. 1982), red pigment-concentrating h o r m o n e ( R P C H ; Mangerich et al. 1986) and pigment-dispersing h o r m o n e ( P D H ; Mangerich et al. 1987). In the XO, M I H - i m m u n o r e a c t i v e perikarya are intermingled with C H H perikarya although sometimes tending to cluster in a peripheral region of the XO. It was notable that C H H cells o u t n u m b e r M I H cells by a b o u t 2:1 in all species examined. With the possible exception of U. pugilator, both cell types are morphologically indistinguishable (size approximately 40 ~tm) and correspond to cell type 5 in C. maenas (Smith and Naylor 1972). Since co-localisation of immunoreactivity was never observed, it is highly unlikely that M I H and C H H are de-
rived from a c o m m o n precursor. Indeed, Stuenkel (1986) has recently demonstrated that C H H and a so-called "peptide H " are the sole products of a 14 K d a precursor peptide in the XO-SG system of Cardisoma carnifex. Since the Hpeptide does not possess cysteine unlike M I H (Webster and Keller 1986), and undergoes further modification and cleavage to make several smaller neuropeptides (Newcomb et al. 1985), the possibility that C H H and M I H are post-translational products of a precursor seems remote. The present direct demonstration of M I H in perikarya of the XO confirms very early observations by Passano (1953) who demonstrated, by ablation experiments, that the XO is the likely source of a moult-inhibiting principle in the eyestalk. By use of classical stains for neurosecretory material, D u r a n d (1956) found that the stainability of a certain cell type (Type 2) in the XO Oreoneetes limosus changed in relation to the moulting cycle, which led him to conclude that this cell type produces MIH. Rehm (1959) using conventional histochemical methods to describe axonal types in the SG of C. maenas observed that two cysteine-containing axon types (A and B) exhibited moult cycle-related changes in tinctorial affinity reminiscent of reduced secretory activity during premoult. Since both C H H and M I H contain cysteine (Keller and W u n d e r e r 1978; Webster and Keller 1986), this observation might suggest that the A and B axon types of Rehm (1959) were identical with those containing C H H and MIH. In the present study no striking moult cycle-related changes in the immunostainability of the SG for M I H and C H H were observed. However, in premoult C. maenas (stage D2) the MIH-immunopositive perikarya consistently exhibited faint immunostaining. Although this does not necessarily indicate an inactive state, we are inclined to interprete it in this way in the present context. It is tempting to speculate upon this p h e n o m e n o n in the light of the accepted paradigm of moult control, which postulates that during premoult, M I H synthesis or release is reduced. It should, however, be noted that in premoult animals, immunostaining of the C H H perikarya was also reduced to a certain extent. Whilst we have previously suggested that C H H may have some moult-controlling function in view of its ability to repress
Fig. 4 a-k. MIH- and CHH-immunoreactivityin Liocarcinus puber and Cancer pagurus, a MIH-immunoreactiveaxon profiles and terminals in the SG of L. puber. Note crossing of incoming axons, x 180. b CHH-immunoreactivity in peripherally arranged Herring body-like dilatations and in finer endings close to the central hemolymph lacuna in the SG of L. puber, x 120. e Cross section through MIHimmunoreactive axons in the XO-SG tract of L. puber arranged around putative CHH axons, x 490. d CHH-immunoreactive perikarya (two darkly and seven lighter stained ones) in the X-organ (XO) with axons and collaterals in the medulla terminalis (MT) of L. puber, x 180. e MIH-immunoreactive perikarya, axons and collaterals in the XO of L. puber, x230. f MIH-immunoreactive axon profiles and terminals in the SG of C. pagurus, x 150. g CHH-immunoreactive axon profiles and terminals in the SG of C. pagurus. x 180, h Cross section through MIH-immunoreactive axons of the XO-SG tract (left) around putative CHH axons and (right) collaterals in the MT of C. pagurus, x 540. i CHH-immunoreactive perikarya in the XO and part of the XO-SG tract in C. pagurus, x 260. k MIH-immunoreactive perikarya in the XO with axons and collaterals in C. pagurus, x 220. For other unexplained abbreviations, see Fig. 2 Fig. 5a-k. MIH- and CHH-immunoreactivity in Uca pugilator and Maja squinado, a MIH-immunoreactive axon profiles and terminals in the SG of U. pugilator. Note Herring body-like dilatations (arrowheads) and peripheral axons (arrows) in the XO-SG tract, x 340. b CHH-immunoreactive axon profiles and terminals in the SG of U. pugilator, x 310. e Cross section through CHH-immunoreactive axons of the XO-SG tract in the MT of U. pugilator, x 1340. d MIH-immunoreactive XO-perikarya and part of the XO-SG tract with peripherally arranged axons (arrows) in the MT of U. pugilator, x 350. e CHH-immunoreactive XO-perikarya and part of the XO-SG tract (arrows) in U. pugilator, x 360. f MIH-immunoreactive axon profiles and endings in the SG of Maja squinado, x 140. g CHH-immunoreactive axon profiles and endings in the SG of M. squinado. Note entering tract and large peripheral immunoreactive axon profiles. • 150. h Cross section through the CHH-immunoreactive axon bundle of the XO-SG tract in M. squinado. Arrow's indicate collaterals, x 490. i MIH-immunoreactive XO-perikarya (arrows) of M. squinado, which stain very faintly and have a granular cytoplasm, x 180. k CHH-immunoreactive XO-perikarya of M. squinado giving rise to the axons of the tract. Note unstained perikarya of the same morphological type (arrows). x 260. For other abbreviations, see Fig. 2
11 ecdysteroid synthesis in vitro, albeit at much higher concentrations than M I H (Webster and Keller 1986), we feel it is as yet p r e m a t u r e to speculate on this subject until m o r e detailed w o r k is completed. A branching plexus o f fine collateral axons distal to the X O consisting o f discrete MIH'- and C H H - i m m u n o r e a c tive elements was seen in all species examined. W i t h regard to the C H H collaterals, it has been suggested that they receive b o t h peptidergic and aminergic inputs (Gorgels-Kallen a n d Van Herp, unpublished work). In C. maenas, Jaros et al. (1985) have observed enkephalin-like immunoreactive fibres in a position c o m p a r a b l e to the area o f branching collaterals described in this study. Thus, it would seem not unlikely that a similar situation exists with regard to the M I H collateral system. In the X O - S G tract, the M I H axons are arranged circumferentially, enclosing the C H H axons. This appears striking in view o f the fact that the M I H p e r i k a r y a do not form a distinct subgroup in the XO but are intermingled with the C H H perikarya. The functional significance, if any, o f this arrangement is not clear. The M I H axons are also completely separate from the tract that transports R P C H and runs alongside the main C H H / M I H tract (Mangerich et al. 1986). In the SG, the M I H secretory terminals were smaller than those containing C H H , and fewer Herring body-like profiles were seen. This observation is in agreement with the H P L C profiles of the S G neuropeptides from which it is evident that M I H is less a b u n d a n t than C H H . One o f the most striking features of C H H is its speciesor systematic-group specificity (for review see Keller et al. 1985), which is thought to reflect a rather rapid molecular evolution of this peptide. Whilst immunocytochemical evidence alone cannot assess this p h e n o m e n o n quantitatively, the results for U. p u g i l a t o r and M . squinado, in which faint staining o f M I H structures was observed even at relatively high concentrations o f antiserum against M I H of C. m a e n as, might suggest a similar rapid evolution o f the neuropeptide in decapods. However, M . squinado and other oxyrhynchan crabs undergo " d e t e r m i n a t e g r o w t h " (Hartnoll 1982) as a result o f degeneration of the Y - o r g a n after the puberty moult, precipitating a terminal anecdysis (Carlisle 1957). It is possible that in such animals synthesis o f M I H is m a r k edly reduced as was suggested by this author. Clearly, further studies are necessary to elucidate this problem. Acknowledgments. The authors wish to thank the following persons
for assistance in obtaining the crabs used in this study: Dr. M. Fonds, NIOZ, Texel, The Netherlands; Cynthia A. Zahnow, University of West Florida, Pensacola; Prof. Y. Le Gal, Concarneau, France. To the latter we are also grateful for generous hospitality at the Laboratoire de Biologic Marine of the Coll+ge de France at Concarneau. We also wish to thank Dr. G. Kegel, Bonn, FRG, for amino acid analyses, and G. Kummer, S. Mangerich and H.-P. Orth (all in Bonn) for their help in collecting sinus glands and with radioimmunoassays. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Ke 206/7-4) to R.K. and by a Royal Society European Fellowship and Royal Society 1983 University Research Fellowship to S.G.W, References Boer HH, Schot LPC, Roubos EW, TerMaat A, Lodder JC, Reichelt D, Swaab DF (1979) ACTH-like immunoreactivity in two
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